title: A high-throughput microfluidic dental plaque biofilm system to visualize and quantify the effect of antimicrobials.
abstract: OBJECTIVE
  Few model systems are amenable to developing multi-species biofilms in parallel under environmentally germane conditions. This is a problem when evaluating the potential real-world effectiveness of antimicrobials in the laboratory. One such antimicrobial is cetylpyridinium chloride (CPC), which is used in numerous over-the-counter oral healthcare products. The aim of this work was to develop a high-throughput microfluidic system that is combined with a confocal laser scanning microscope (CLSM) to quantitatively evaluate the effectiveness of CPC against oral multi-species biofilms grown in human saliva.
  
  
  METHODS
  Twenty-four-channel BioFlux microfluidic plates were inoculated with pooled human saliva and fed filter-sterilized saliva for 20 h at 37°C. The bacterial diversity of the biofilms was evaluated by bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). The antimicrobial/anti-biofilm effect of CPC (0.5%-0.001% w/v) was examined using Live/Dead stain, CLSM and 3D imaging software.
  
  
  RESULTS
  The analysis of biofilms by bTEFAP demonstrated that they contained genera typically found in human dental plaque. These included Aggregatibacter, Fusobacterium, Neisseria, Porphyromonas, Streptococcus and Veillonella. Using Live/Dead stain, clear gradations in killing were observed when the biofilms were treated with CPC between 0.5% and 0.001% w/v. At 0.5% (w/v) CPC, 90% of the total signal was from dead/damaged cells. Below this concentration range, less killing was observed. In the 0.5%-0.05% (w/v) range CPC penetration/killing was greatest and biofilm thickness was significantly reduced.
  
  
  CONCLUSIONS
  This work demonstrates the utility of a high-throughput microfluidic-CLSM system to grow multi-species oral biofilms, which are compositionally similar to naturally occurring biofilms, to assess the effectiveness of antimicrobials.
authors.affiliation: Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, MI 48109-2029, USA.
authors.forename: William C
authors.initials: WC
authors.lastname: Nance
authors.forename: Scot E
authors.initials: SE
authors.lastname: Dowd
authors.forename: Derek
authors.initials: D
authors.lastname: Samarian
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Chludzinski
authors.forename: Joseph
authors.initials: J
authors.lastname: Delli
authors.forename: John
authors.initials: J
authors.lastname: Battista
authors.forename: Alexander H
authors.initials: AH
authors.lastname: Rickard
mesh_terms: D000328:Adult; D000890:Anti-Infective Agents / Q000494:pharmacology*; D018441:Biofilms / Q000187:drug effects* / Q000254:growth & development*; D002594:Cetylpyridinium / Q000494:pharmacology*; D003773:Dental Plaque / Q000382:microbiology*; D006801:Humans; D059013:Microbial Consortia / Q000187:drug effects*; D044085:Microfluidics / Q000379:methods*; D018613:Microscopy, Confocal; D012463:Saliva / Q000378:metabolism / Q000382:microbiology
chemical_list: D000890:Anti-Infective Agents; D002594:Cetylpyridinium
keywords: Live/Dead staining; confocal scanning laser microscopy; microfluidics; multi-species biofilm; pyrosequencing